INTRODUCTION:

Herbal medicine also known as phytotherapy is the science of using herbal remedies to treat various sickness. It covers all the aspects of herbal medicine, that is, plants with powerful actions to those with gentle actions. Currently, people have come to know of the side effects of synthetic drugs, thus leading to an increased development of natural products as treatment for ailments [1]. This lead to an increase in the curiosity of the scientists and various pharmaceutical industries to develop new herbal medicine for various diseases with decreased side effects [2]. India, a country with highest number of plants with medicinal and therapeutic importance became a target for these pharmaceutical industries to export these plants for their plan development. Thus, leading to an increase in the export business of India [3].

Epilepsy is a major neurological disorder in which a person suffers from recurrent unprovoked seizures. Epilepsy being a common disorder of the CNS was found to affect 5% of the world population [1]. Seizure is the characteristic feature in epilepsy and is associated with disordered and rhythmic high frequency discharge of impulses by a group of neurons in the brain. The current therapy for epilepsy with the modern synthetic antiepileptic drugs were found to be having certain dose-related side effects and toxicity [4]. Herbs were claimed on having better antiepileptic effects. Hence phytochemicals were identified from various traditional medicinal plants, thus presenting an exciting opportunity for the development of new types of therapeutics.

Antiepileptic drugs (AED’s) protects the body against seizures by interaction with various cellular targets. They suppress the abnormal hyperactivity of the brain cells. But currently, certain antiepileptic drugs have been found to fail to provide adequate control of epileptic seizures. This has stimulated researchers to develop drugs with better activity. Hence, the medicinal plants were also considered to be an important source in the development of drugs with this biological activity.

Annonareticulata Linn. belongs to family Annonaceae. The plant is most commonly also known as Ramphal, Bullock's heart and Custard apple. Nearly about 119 different species of the Annona genus (Annonaceae) are identified, most of them being shrubs and trees [5]. In India, it is widely cultivated and naturalized as a fruit consuming plant and deciduous tree. In old system of medicines, this plant was reported to have pharmacological activities such as antifungal, anticancer, spasmolytic, anticonvulsant, antimalarial, anthelmintic and anti-syphilitic [6]. The present study is designed to establish the antiepileptic activity of leaves of Annona reticulata to support the traditional usefulness of the leaves extract in epilepsy.

MATERIALS AND METHODS:

Plant Material:

The leaves of Annona reticulata were collected in the month of April-May from Mangalore, Karnataka. The botanical identity of the leaves of the plant was verified and authenticated by Dr. K.V. Nagalakshamma, HOD, Department of Botany, St. Aloysius College, Mangalore. A voucher specimen (No. 16PC007) was deposited in N.G.S.M Institute of Pharmaceutical Sciences, Paneer, Deralakatte, Mangalore.

Preparation of Extracts:

The leaves were collected from Mangalore, Karnataka, During May 2017. The leaves were cleaned from adhering soil, dust and other material and then it was dried under the shade for 30 days. The dried leaves were cut into small pieces and finely powdered (5Kg) by using a dry grinder. The powdered leaves were subjected to cold maceration extraction using ethanol as the solvent [7]. They were exhaustively extracted by maceration with ethanol for three days, after three days, the ethanol layer was decanted off. The process was repeated thrice. The solvent obtained after decantation was distilled off and the concentrate was evaporated to a syrup like consistency by maintaining the required temperature conditions and then was evaporated to dryness (545g) on an open water bath at room temperature to a dark green solid mass. It was stored in a desicator.

Animals:

Healthy young adult Albino rats of laboratory strains were employed. Each animal selected for the dosing were in between 8 and 12 weeks old. The animals were bred under the standard laboratory conditions, the temperature in the animal room was kept at 22⁰C (±3⁰ C) and the relative humidity was at 50% to 60%. The animals were kept for 5 days prior to the studies for acclimatization to the laboratory conditions and fasted overnight ad libitum prior dosing of above extract. All the experiments were carried out between 0900 to 1500 hrs [8].

Assessment of antiepileptic activity:

Maximal Electroshock Shock Induced Seizures:

The fasted animals divided into five groups of six rats were selected for the study. Group III, IV and V received the test drug at three different dose level (100mg/kg, 200mg/kg and 400mg/kg body weight) orally 60 min prior to induction of MES. Group II received the standard drug (Phenytoin, 25mg/Kg body weight, i.p) 30 min before the induction of MES, Group I received the vehicle (0.6% Sodium Carboxy Methyl Cellulose) served as control. The Animals were subjected to MES at 150 mA, 60 Hz for 0.2 sec through pinnal electrodes at 60 min after vehicle/drug administration [9]. In the electrically induced convulsions, the animal were manually restrained and released immediately to permit observation. MES resulted in tonic hind limb extension, the duration of which was measured in seconds. Animals were observed for various phases of MES seizures like tonic hind limb flexion, tonic hind limb extensor and tonic clonic phase. The decrease or abolition of the duration of the extensor phase was considered an index of anti epileptic activity [10].

Pentylenetetrazole Induced Seizures:

The fasted animals divided into five groups of six rats were selected for the study. Group III, IV and V received the test drug at three different dose level (100mg/kg, 200mg/kg and 400mg/kg body weight) orally one hour prior to the administration of PTZ. Group II received the standard drug (Diazepam. 4mg/Kg body weight, i.p) 30 min prior to the PTZ treatment, Group I received the vehicle (0.6% Sodium Carboxy Methyl Cellulose) served as control. One hour after the administration of the compound, vehicle and standard drug, PTZ (80mg/Kg body weight, i.p) was administered to induce clonic convulsions. All animals were observed for a period of 30 mins after the PTZ administration in their isolation cages. The experiment was assessed by its ability to delay the onset of clonic convulsions. The protection against the PTZ induced convulsions was measured [9].

Statistical Analysis:

The Data values are presented as Mean±SEM, and the statistical significance between the treated and control group were analysed using one way ANOVA, followed by the Dunnet’s test where p<0.05 was considered statistically significant.

RESULTS:

Acute Toxicity Study:

The acute toxicity studies of the methanolic and aqeous extract of Annonareticulata did not show any sign of toxicity up to 2000mg/kg body weight. Based on this, the dose of the extract selected for the study was 400mg/kg (high dose), 200mg/kg (medium dose) and 100mg/kg (low dose).

Maximal Electroshock Model

In the present study, It was found that the methanolic and aqeous extract of the leaves of Annonareticulata on oral administration were found to decrease the duration of the tonic hind limb extension. The tonic hind extension phase of the rat at the dose of 200mg/kg and 400mg/kg was found to be 12.40±0.324sec and 08.23±0.621sec for methanolic extract and 15.51±0.571sec and 09.37±0.736sec for aqeous extract, significant than that of the control group. The percentage of convulsion was found to be 17% and 33.3% at 400mg/kg and the percentage of mortality was found to be 0 at 400mg/kg. The observations are recorded in Table 5.

Pentylenetetrazole Model:

In the present study, It was found that when the rats were treated with the methanolic and aqeousextract by oral administration, they were found to show good antiepileptic activity by delaying the onset of convulsions. The onset of Jerks, Straubtail and Clonus was found to be delayed in the case of 200mg/kg and 400mg/kg. The methanolic extract of 400mg/kg dose was found to show 100% protection to the rats, where as in aqeous extract at 400mg/kg was found to show OJ at 147.55±4.26, OS at 171.39±5.36 and OC at 221.47±4.288sec. The observations are recorded in Table 6.

Table 1: Effect of Methanolic and Aqeous extract of the leaves of Annonareticulata on seizures induced by Maximal Electroshock in Rats

Treatment	Dose (mg/kg)	Tonic Hind Limb Extension Phase in sec (Mean±SEM)	No of Animals Convulsed / No of Animals Used	% C	% M
Control	Vehicle	18.72±0.872	6/6	100	83.33
Phenytoin	25	02±0.33	1/6	17	00
MEAR	100	19.56±0.516	6/6	100	50
MEAR	200	12.40±0.324*	3/6	50	17
MEAR	400	08.23±0.621*	1/6	17	00
AEAR	100	18.66±0.843	6/6	100	33.3
AEAR	200	15.51±0.571*	4/6	66.67	17
AEAR	400	09.37±0.736*	2/6	33.3	00

*results are expressed as Mean±SEM; significance at P<0.05* as compared to control.

MEAR – Methanolic extract of leaves of Annona reticulata, AEAR – Aqeous extract of leaves of Annona reticulate, %C – Percentage Convulsions, %M - Percentage Mortality.

Table2: Effect of Methanolic and Aqeous extract of the leaves of Annona reticulata on seizures induced by Pentylenetetrazole in Rats.

Treatment	Dose (mg/kg)	Observation in Seconds (Mean ± SEM)			% C
Treatment	Dose (mg/kg)	OJ	OS	OC	% C
Control	Vehicle	42.21±2.531	54.16±1.325	67.271±1.913	100
Diazepam	5	236.14±6.392	341.67±7.152	No	00
MEAR	100	50.24±1.35	69.32±3.24	82.43±2.42	83.3
MEAR	200	77.13±2.17	86.134±2.13	97.15±3.18	50
MEAR	400	No	No	No	00
AEAR	100	57.352±2.13	61.45±3.16	87.56±2.35	100
AEAR	200	109.34±3.26	121.26±3.87	138.54±1.876	67.6
AEAR	400	147.55±4.26	171.39±5.36	221.47±4.288	17

*results are expressed as Mean±SEM; significance at P<0.05* as compared to control. MEAR – Methanolic extract of leaves of Annonareticulata, AEAR – Aqeous extract of leaves of Annonareticulata, OJ – Onset of Jerks, OS – Onset of Straub’s tail, OC – Onset of Clonus.

DISCUSSION:

The present study revealed that the methanolic and aqeous extract of the leaves of Annona reticulata L. has shown anticonvulsant activity against seizure induced by MES and PTZ Model. The results suggested that the Annona reticulata leaf extract was effective in blocking both the tonic clonic and Myoclonic seizures. According to ancient medicine, epilepsy patients were asked to eat one leaf of Annona squamosa for relief from the epileptic attacks. The leaves extract of Annona squamosa were found to protect rats from the convulsive attacks. However there was no such study done on the plant of the same genus Annona reticulataand its phytochemical constituents. On this basis, the leaf of Annona reticulata were evaluated for the same activity.

CONCLUSION:

The evaluation of the antiepileptic activity was done by MES induced convulsion and PTZ Induced convulsion. The aqeous and methanolic extract of leaves of Annona reticulata was found to show maximum protection to the rats from the seizures induced by both MES and PTZ Methods. In the case of MES induced convulsion, both the extracts delayed the duration of HLTE and showed activity almost comparable to Phenytoin. In the case of PTZ induced convulsion, both the extracts delayed the onset of clonus and straubs tail and showed activity comparable to Diazepam. The leaf extract at the dose of 400mg/kg showed the highest activity, and was found to be better than the standard. The rats showed no signs of convulsion and were completely protected. The % Mortality and the % Convulsion of the rats was found to decrease with a increase in the dose of the leaf extract administered orally. The statistical representation of the values obtained for the aqeous and methanolic extract at the dose of 200mg/kg and 400mg/kg was found to be significant. This leads to the conclusion that the leaves of Annona reticulata possesses significant antiepileptic activity.

ACKNOWLEDGEMENT:

Authors are thankful to Nitte (Deemed to be University) and N.G.S.M.Institute of Pharmaceutical Sciences, Mangalore, for providing all necessary facilities to conduct the experiment.

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Received on 31.08.2018 Modified on 29.09.2018

Research J. Pharm. and Tech 2019; 12(1): 241-244.

DOI: 10.5958/0974-360X.2019.00045.3